Date Published: June 01, 2016
Publisher: International Union of Crystallography
Author(s): Helen Mary Ginn, David Ian Stuart.
An iterative method of map recovery for perfectly merohedrally twinned crystals in the presence of noncrystallographic symmetry is reimplemented and released and an independent metric of success is provided.
Biological crystals are occasionally, but not uncommonly, subject to perfect or imperfect merohedral twinning (Yeates, 1997 ▸; Yeates & Fam, 1999 ▸), where unit cells or mosaic domains are randomly distributed into two or more orientations without affecting the crystal lattice. This is particularly common in virus-capsid crystallography, where spherical capsids can rotate without significantly altering the minimal crystal contacts (Lerch et al., 2009 ▸). For some crystal systems, twinning can be minimized or avoided by altering the concentration of nuclei for crystallization (Chayen & Saridakis, 2008 ▸) or deliberately choosing crystals that grow at a slower rate (Borshchevskiy et al., 2009 ▸). When the merohedral twinning fraction is measurably below 0.5, data recovery is comparatively easier and quite frequently allows structure solution by de novo methods. For molecular-replacement solutions there are a large number of examples (Breyer et al., 1999 ▸; Igarashi et al., 1997 ▸; Carr et al., 1996 ▸; Luecke et al., 1998 ▸; Chandra et al., 1999 ▸; Contreras-Martel et al., 2001 ▸). For anomalous phasing, notable examples include interleukin-1 (Rudolph et al., 2003 ▸) and a selenomethionine variant of the capsid-stabilizing protein of bacteriophage λ, gpD (Yang et al., 2000 ▸), which were both solved by multiwavelength anomalous dispersion (MAD). Twinned crystals of bilirubin oxidase with a twin fraction of 0.487 were solved by SAD (Mizutani et al., 2010 ▸). However, perfect merohedral twinning is often more challenging to overcome, and most commonly requires molecular replacement to solve the structure (Chandra et al., 1999 ▸; Redinbo & Yeates, 1993 ▸; Lea & Stuart, 1995 ▸). However, the gpD structure has been solved by SAD, where the data were averaged to emulate a twinning fraction of 0.5 (Dauter, 2003 ▸). Twinning presents itself as a higher symmetry space group and may be more difficult to detect immediately if analysis of the crystal-packing density is not unambiguous. However, it causes an enrichment of mid-intensity reflections owing to the superposition of the two crystal orientations, where combinations of two low-intensity or two high-intensity reflections are less common. In fact, it is common for proteins to be submitted to the PDB with their partially twinned nature going unnoticed (Lebedev et al., 2006 ▸). Programs such as TRUNCATE, which is part of the CCP4 suite, now test for this distorted intensity distribution as standard (Winn et al., 2011 ▸).
Reflections for O1M and artifically twinned O1BFS were transformed into real space. These electron-density maps were averaged using fivefold NCS and scaled according to resolution shell using only singlet reflections for a total of 20 cycles. R factors and correlation coefficients were measured between observed twinned data and partially detwinned data, for both the whole set of reflections (Rall, CCall) and the singlet subset (Rsinglets, CCsinglets), at each stage of the cycle (R factors are shown in Fig. 2 ▸, including the result from incorrect NCS operators). The singlet reflections are treated specially, rather than setting them equal to the amplitudes in the twinned data set: they are only scaled globally. This allows them to be used as a measure of success by tracking their agreement with the original amplitudes over several rounds of fivefold NCS averaging, as they are unaffected by twinning.
The data analysis suggests that the deconvolution of twinned crystals with rotational NCS, which is distinct from the symmetry of the twinning operators, is successful. The control data set used here also suggests that the error can be reduced to within 6% of the error already present during data collection. The success of the deconvolution process can be measured by separately processing and tracking the R factor for singlet reflections only, and is verified visually by comparing the electron density. Furthermore, this method will be highly applicable to other virus crystal structures that possess high rotational NCS and a high propensity for twinning owing to their pseudo-spherical nature, as well as other twinned structures that exhibit similar NCS and twinning-operator relationships. This could be applied to the six point groups that support true merohedral twinning (Yeates, 1997 ▸). Tables of space groups that can lead to this problem, point groups and possible twin operators have been discussed (Chandra et al., 1999 ▸). The source code for solving hemihedral twinning, written primarily in C++, is available along with an example structure and script (http://github.com/helenginn/deconvolute). It requires the CCP4 tools to be installed, but provides the other external Fortran tools required to run the program. Compilation has been tested on the GCC compiler v.4.4.7.